The present invention relates to labels, preferably metal-containing labels for use in assays. In particular, the invention relates to metal labels having ligands substituted with hydrophilic and/or charged groups that prevent the non-specific binding (xe2x80x9cNSBxe2x80x9d) of labeled substances to other materials. The invention also relates to methods for conducting assays, preferably luminescence assays, that use these labels and to kits and compositions containing these labels.
Documents cited in this application relate to the state-of-the-art to which this invention pertains. The disclosures of each of these references are incorporated herein by reference.
Electrochemiluminescence (ECL) detection has become an important analytical technique and has been applied in general analysis and diagnostic procedures. Electrochemiluminescence involves electrogenerated species and the emission of light. For example, electrochemiluminescence may involve luminescence generated by a process in which one or more reactants are generated electrochemically and undergo one or more chemical reactions to produce species that emits light, preferably repeatedly.
In practice, most ECL-based assays involve the use of electrochemiluminescent compounds as labels. The presence of a labeled substance or the participation of a labeled substance in a binding reaction is determined via detection of electrochemiluminescence from the ECL label. Assays for analytes based on the use of labeled binding reagents specific for an analyte of interest may be homogenous in nature (see U.S. Pat. No. 5,591,581 and Published PCT Application WO87/06706) or may involve binding reactions occurring on a solid phase such as a magnetic particle (see U.S. Pat. No. 5,705,402) or an electrode surface (see U.S. Pat. Nos. 6,066,448 and 6,207,369 and Published PCT Application WO98/12539).
An important class of ECL labels is organometallic complexes of ruthenium, osmium or rhenium having one or more polydentate heterocyclic nitrogen containing ligands (e.g., bipyridine, phenanthroline, bipyrazine, bipyrimidine, etc., or substituted derivatives thereof) such as those described in U.S. Pat. Nos. 5,310,687; 5,597,910; and 5,591,581 and Published PCT Application WO87/06706. These types of labels (in particular, labels based on tris-bipyridyl ruthenium complexes) have found considerable use because of their stability and the efficiency at which they produce ECL.
In commercial ECL instrumentation, ECL from these labels is typically produced by oxidizing the labels in the presence of an ECL coreactant, such as tripropylamine. The ECL coreactant is also oxidized at the electrode to produce a strong reductant (see, e.g., U.S. Pat. No. 5,846,485). The highly energetic reaction of the reductant and the oxidized label leads to reduction and excitation of the label to a luminescent excited state. Emission of a photon regenerates the label in its original state and allows for detection of the label.
Electrochemiluminescence is an extremely sensitive detection technique. The sensitivity of the detection technique is often, however, not the determining factor for the sensitivity of a particular assay. In assays that involve the specific binding interaction between a labeled binding reagent and a binding partner (e.g., an analyte), the sensitivity is often limited by the background signal resulting from the non-specific binding (NSB) of the labeled binding reagent with substances other than the binding partner, e.g., other components of crude samples, other assay reagents, or in the case of solid phase binding assays, the solid phase itself. In some cases, NSB may also lead to a lowering of signals through loss of reagent on the surfaces of containers, pipettes, etc. While ECL labels generally have better NSB properties than other classes of labels, under certain conditions NSB may be a limiting factor in assay sensitivity. This occurs, for example, in i) ECL assays using labeled binding reagents, where the binding reagent itself exhibits high levels of NSB; ii) ECL assays using binding reagents labeled with large numbers of labels and iii) ECL assays carried out using low concentrations of blockers of NSB (such as blocking proteins or detergents) or carried out in the absence of such blockers.
The present invention relates to substituted bipyridines and phenanthrolines having at least one and preferably two substituents, the substituents comprising negatively charged groups, preferably sulfate or sulfonate groups. These substituted bipyridines and phenanthrolines, when present as a ligand in a metal complex, reduce the NSB of the complex relative to analogous unsubstituted bipyridines or phenanthrolines. In addition, the present invention relates to organometallic complexes comprising such ligands and labeled assay reagents comprising such organometallic complexes.
The present invention also relates to luminescent metal complexes having the structure
ML1L22
wherein
M is Os or Ru;
L1 is L2 as described below or a substituted bipyridine or phenanthroline ligand having at least one substituent that is covalently linked to i) a biological material and/or an assay reagent useful in an assay or ii) a moiety that can participate in a reaction with a biological material and/or an assay reagent useful in an assay so as to form such a covalent linkage; and
L2 is a substituted bipyridine or phenanthroline ligand that comprises a negatively charged group, preferably a sulfate or sulfonate group, said group acting to reduce the NSB of the complex relative the analogous complex in which L2 is unsubstituted bipyridine or phenanthroline. Alternatively, L2 is a substituted bipyridine or phenanthroline ligand that comprises a neutral hydrophilic group, preferably a hydroxyl group or a carboxamide, or a positively charged group, preferably, a guanidinium group.
The present invention also relates to luminescent metal complexes having the structure
ML1L22
wherein
M is Os or Ru;
L2 is a metal ligand selected from the group consisting of: 
wherein,
T is a linker group comprising an alkyl, alkenyl, alkynyl or phenyl linker, or a combination thereof, having, optionally, one or more chain carbons substituted by a heteroatom;
Z is xe2x80x94SO3xe2x88x92, xe2x80x94SO3H, xe2x80x94OSO3xe2x88x92, xe2x80x94OSO3H, xe2x80x94PO32xe2x88x92, xe2x80x94PO3Hxe2x88x92, xe2x80x94PO3H2, xe2x80x94OPO32xe2x88x92, xe2x80x94OPO3Hxe2x88x92, xe2x80x94OPO3H2, xe2x80x94OP(R)O2xe2x88x92, xe2x80x94OP(R)O2H, xe2x80x94[NHC(NH2)2]+, or xe2x80x94NHC(NH)NH2; and
R is alkyl; and
L1 is a substituted bipyridine or phenanthroline ligand having at least one substituent that is covalently linked to i) a biological material and/or a binding reagent useful in an assay or ii) a moiety that can participate in a reaction with a biological material and/or a binding reagent useful in an assay so as to form such a covalent linkage; and L1 is, preferably, selected from the group consisting of 
wherein,
X is a linker group comprising an alkyl, alkenyl, alkynyl or phenyl linker, or a combination thereof, having, optionally, one or more chain carbons substituted by a heteroatom;
Y is H or alkyl and
W is a functional group that is linked to a biological molecule, binding reagent, enzyme substrate or other assay reagent or W is a functional group that when present on the ligand is suitable for conjugating the ligand to a biological material, binding reagent, enzyme substrate or other assay reagent.
The present invention also relates to labeled materials having one or more metal complexes of the invention, preferably luminescent metal complexes, linked to a material. In one embodiment, the invention relates to labeled materials having the structure [A]i[B]j, wherein A is a luminescent metal complex of the invention, B is a substance (preferably a biological material and/or an assay reagent useful in an assay) covalently linked to one or more A, i is an integer greater than zero and j is an integer greater than zero (preferably, one). Preferably, A is a metal complex with the structure ML1L22 as described above and A and B are covalently linked via a functional group on L1.
The present invention also relates to the use of the luminescent metal complexes of the invention for the generation of luminescence. The complexes may be used in luminescence-based assays such as assays based on the measurement of photoluminescence intensity, time resolved photoluminescence, luminescence energy transfer, luminescence quenching, luminescence lifetime, luminescence polarization, chemiluminescence or, preferably, electrochemiluminescence. The invention also includes the use of complexes of the invention in non-luminescent assays such as electrochemical assays (i.e., assays involving the measurement of current or voltage associated with the oxidation or reduction of the complexes) including electrochemical assays that use the metal complex as a redox label and electrochemical assays that use the metal complex as a redox mediator for measuring the reduction or oxidation of an analyte (e.g., DNA). Preferably, the use of the metal complexes of the invention leads to improved assay performance through low non-specific binding of the complex relative to analogous complexes not presenting low NSB functional groups.
The present invention also relates to methods of measuring the labeled materials of the invention comprising the steps of i) contacting the labeled materials with a binding reagent and, optionally, a solid phase support; ii) forming a binding complex comprising the binding reagent, the labeled materials, and, optionally, the solid phase support; iii) inducing the labeled materials to produce a signal, preferably, luminescence, more preferably ECL and iv) measuring the signal so as to measure the luminescent metal complex. Preferably, the use of the metal complexes of the invention leads to improved assay performance through low non-specific binding of the metal complex relative to analogous metal complexes not presenting low NSB functional groups.
The present invention also relates to methods of measuring an analyte in a sample comprising the steps of i) contacting the sample with a labeled binding reagent and optionally a solid phase support; ii) forming a binding complex comprising the binding reagent, the analyte and, optionally, the solid phase support; iii) inducing labels in the labeled binding reagent to produce a signal, preferably, luminescence, more preferably ECL and iv) measuring the signal so as to measure the analyte in the sample; wherein the labeled binding reagent comprises one or more of the low NSB labels described above covalently linked to a binding reagent specific for the analyte. Preferably, the use of the labels of the invention leads to improved assay performance through low non-specific binding of the labels relative to analogous labels not presenting low NSB functional groups.
The present invention also relates to methods of measuring an analyte in a sample comprising the steps of i) contacting the sample with a labeled analog of the analyte, a binding reagent and, optionally, a solid phase support; ii) forming a binding complex comprising the labeled analog of the analyte, the binding reagent and, optionally, the solid phase support; iii) inducing labels in the labeled analog of the analyte to produce a signal, preferably, luminescence, more preferably ECL and iv) measuring the signal so as to measure the analyte in the sample; wherein the labeled analog of the analyte comprises one or more of the low NSB labels described above covalently linked to an analog of the analyte, and wherein said analog of the analyte competes with the analyte for binding to the binding reagent. Preferably, the use of the labels of the invention leads to improved assay performance through low non-specific binding of the complex relative to analogous labels not presenting low NSB functional groups.
The present invention also relates to methods of measuring an analyte or a chemical or biological activity in a sample comprising the steps of i) contacting a sample containing the analyte or the chemical or biological activity (or a sample containing substrates or products of the activity) with a low NSB metal complex of the invention; ii) inducing the metal complex to produce a signal, preferably luminescence, more preferably electrochemiluminescence and iii) measuring the signal so as to detect or measure the chemical or biological activity.
The present invention also relates to methods of improving existing assays employing metal complexes by replacing the metal complexes or ligands on the metal complexes with the low NSB labels or ligands of the invention.
The invention further relates to kits and compositions containing the low NSB ligands and metal complexes of the invention.